Control of gene expression is mediated partly by recruitment of transcriptional factors to the promoter elements. Recruitment of these factors is further regulated by the chromatin structure surrounding the promoter region. If the chromatin structure is in an open conformation, the promoter element is accessible to the transcriptional factors but if it is in a closed conformation, the promoter element is inaccessible. Using a novel yeast strategy, we cloned a human serine- threonine kinase that appears to convert chromatin from a closed conformation to an open conformation. This kinase is expressed in all tissue types and is localized to the nucleus of human cells. The mechanism by which this kinase opens up the chromatin is currently being investigated. In addition to chromatin opening activity, it appears to respond to DNA damage and may be important for DNA damage response. Once the chromatin is opened, transcriptional factors are recruited to activate or suppress transcription. Although a great deal is known about the transcriptional factors, study of their recruitment would be greatly enhanced by a methodology that allows detection of their recruitment in vivo, a methodology that does not exist yet. Toward this end, we developed a novel methodology PIN*POINT (protein position identification with nuclease tail). In this method, a fusion protein composed of a chosen protein linked to a non-sequence-specific nuclease is expressed in vivo and the binding of the protein to DNA is made detectable by the nuclease-induced cleavage near the binding site. Using PIN*POINT, we have been able to demonstrate that the beta-globin LCR (Locus Control Region) promotes recruitment of transcriptional activator Sp1 to the beta-globin promoter in MEL (murine erythroleukemia) cells. We have also studied recruitment of erythroid specific transcription factor EKLF and chromatin remodelling factor BRG1 using this technique and we find that they are both recruited to the promoter of the beta-globin gene by the beta globin locus control region. In collaboration with Dr. Myung Kim, we are also studying the role of the thyroid hormone receptor and thyroid hormone in recruitment of the general transcription factors. We find that the thyroid hormone receptor, in the absence of thyroid hormone, suppresses the recruitment of Sp1 to the thyroid hormone responsive promoter. In the presence of thyroid hormone, we have been able to show that DNA loops bringing together the thyroid hormone receptor binding element and the promoter. - Understanding transcription and chromatin structure in mammalian gene expression